Compositions and methods of inhibiting gene expression in a lung

ABSTRACT

The present invention provides compositions and methods for delivery to a lung tissue comprising a small interfering RNA (siRNA) capable of inhibiting expression of a gene, and a surfactant. In one aspect, a non-polymeric methods composition comprising a small interfering RNA (siRNA) capable of inhibiting expression of a gene, and a surfactant is disclosed. In other aspects, a method of inhibiting gene expression in a lung of a subject in need thereof and treating bronchopulmonary dysplasia in a lung of a subject also disclosed. The methods comprise administering a therapeutically effective amount of a non-polymeric composition to the lung of the subject, wherein the non-polymeric composition comprises a small interfering RNA (siRNA) capable of inhibiting expression of a gene, and a surfactant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/015,032, filed on Jun. 20, 2014, which is hereby incorporated byreference in its entirety herein.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with government support under HL085103 andHL074195, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Bronchopulmonary dysplasia (BPD) is the most common chronic respiratorydisease in infants and is a devastating condition that disrupts thedevelopmental program of the lung secondary to preterm birth and aninteraction between genetic-environmental factors (hyperoxia, invasivemechanical ventilation and sepsis). Although the definition of BPD hasevolved over the past decade, it is currently defined as the need foroxygen supplementation for 28 days of life and a “physiologic”assessment of the oxygen (O₂) requirement at 36 weeks postmenstrual age.It is estimated that 10,000-15,000 new cases of BPD occur each year inthe United States, and significantly, 97% of all BPD cases occur ininfants with a birth weight less than 1250 grams. Despite many advancesin neonatal ventilation techniques, widespread use of surfactant andantenatal corticosteroids, as well as aggressive fluid management, theincidence of BPD has remained the same or has increased slightly.

Management of BPD takes a considerable toll on health services. Amongpreterm infants, the single costliest complication of hospitalizationduring infancy is BPD, with an average cost per discharge of $116,000.Additionally, BPD is associated with significant pulmonary andneurodevelopmental sequelae that continue to have health ramificationsfor the patient into adulthood. It is thus important to understand thelong-term consequences of BPD, as they are likely to have a significantimpact on treatment and cost and application of health care during thelifetime of those born prematurely.

To date, there is no specific and effective prevention or treatment forBPD. Therefore, a need exists in the art for compositions and methods toprevent and treat BPD in premature babies, infants and children, whichprevent or treat acute lung injury in older children and adults.

SUMMARY OF THE INVENTION

As described herein, the present invention includes compositions andmethods for inhibiting expression of a gene in a lung tissue. In oneaspect, the invention includes a non-polymeric composition comprising asmall interfering RNA (siRNA) capable of inhibiting expression of agene, and a surfactant, wherein the composition is formulated fordelivery to a lung tissue.

In another aspect, the invention includes a method of inhibiting geneexpression in a lung of a subject in need thereof comprisingadministering a therapeutically effective amount of a non-polymericcomposition to the lung of the subject, wherein the non-polymericcomposition comprises a small interfering RNA (siRNA) capable ofinhibiting expression of a gene, and a surfactant.

In yet another aspect, the invention includes a method of treatingbronchopulmonary dysplasia in a lung of a subject comprisingadministering a therapeutically effective amount of a non-polymericcomposition to the lung of the subject, wherein the non-polymericcomposition comprises a small interfering RNA (siRNA) capable ofinhibiting expression of a hyperoxia-induced gene, and a surfactant.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the siRNA comprises an RNA that inhibitsexpression of at least one gene encoding a protein selected from thegroup consisting of C/EBP homologous protein (CHOP), interferon-gamma(IFN-γ), transforming growth factor-beta 1 (TGF-β1), and angiopoietin 2(Ang2). In another embodiment, the siRNA is selected from the groupconsisting of CHOP siRNA, Ang2 siRNA, and anti-sense made against themature miRNA34a sequence. In still another embodiment, the siRNA isselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQID NO:3. In yet another embodiment, the siRNA is an antagomir, such as amiR34a antagomir. In still another embodiment, the siRNA comprises anRNA that inhibits expression of at least one gene encoding ananti-inflammatory protein selected from the group consisting of Sirt1,Bcl2, Ang1, Tie2, Ald, DLL1, Notch 1, Notch 2, CDK4, Cyclin D1, caspase3, caspase 8, caspase 9, Fas, and Fas-L.

In some embodiments of the above aspects or any other aspect of theinvention delineated herein, the surfactant comprises a phospholipid. Inone embodiment, the phospholipid comprises phosphotidylcholine orderivatives thereof.

In another embodiment, the composition is formulated for intranasal orinhalation administration. In one embodiment, the composition isdelivered to alveoli in the lung.

In some embodiments of the above aspects or any other aspect of theinvention delineated herein, the composition further comprises aninhibitor of cox-2.

In one embodiment, the invention includes assessing dysregulatedvascularization in the lung.

In another embodiment, the lung is hyperoxic.

In yet another embodiment, the subject has at least one ofbronchopulmonary dysplasia and hyperoxia-induced cell death in the lung.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing time-course expression of the mature form ofmicro RNA (miR) 34a in lungs of newborn mice exposed to hyperoxia andthe BPD model. C57B16/J newborn mice were exposed to hyperoxia (100% O₂)or room air from post natal day (PN)1-7, and sacrificed at specifiedtime points. The hyperoxia-induced BPD mouse model was generated byexposing newborn mice to hyperoxia from PN1-4, followed by recovery inroom air from PN5 to PN14. Mice were sacrificed and lungs removed forRNA extraction. Estimation of miR34a was done by real-time PCR. BPDcorresponds to PN14.*p<0.05; **p≦0.001.

FIG. 2A is a panel of images showing the absence (left image) orpresence (right image) of the effects of hyperoxia in the lungs of ahyperoxia-induced BPD mouse model.

FIG. 2B is a graph showing the chord length (an indicator of meanalveolar size) in newborn wild type (WT) mice or hyperoxia-induced BPDmice at PN14.

FIG. 3 is a graph showing expression of miR34a in human trachealaspirates. Comparison of miR34a expressions in infants (n=35) withrespiratory distress syndrome (RDS), who did not develop BPD (n=8; whitebar) and who died/developed BPD (n=27; black bar). Airway secretionswere collected from infants who were mechanically ventilated and hadendotracheal tubes in place in the first week of life. After cellseparation, RNA was extracted and the expression of miR34a was measuredusing realtime PCR. Data expressed as mean±standard error of the mean.

FIG. 4 is a panel of images showing in situ hybridization of miR34a(darker staining; 40×) in post-mortem lungs of premature infants atvarious stages of development and/or with RDS/BPD. Post-mortem samplesof lungs were collected from infants with RDS, BPD, and or full-termcontrols of the same postnatal age. The lung samples were used tolocalize and quantify the degree of miR34a expression. Numbers followingRDS labels on top of panels indicate number of days being managed forRDS prior to death. “+” to “++++” at the bottom of each panel provide asemi-quantitative assessment of miR34a expression. Arrows in the100×insets point to Type II alveolar epithelial cells (AEC).

FIG. 5 is a graph showing chord length analysis in newborn WT miceadministered miR34a-antagomir in room air at PN14. The miR34a-antagomirwas administered on PN1 and repeat doses given on PN3 and PN4. Chordlength measurements (an indicator of mean alveolar size) are shown inthe mice lungs administered miR34a-antagomir in RA, at PN14, along withappropriate control groups. *p<0.05 vs. RA; #p<0.05 vs. scrambled. RA:room air; Scrambled: (control) RNA with the same nucleotide compositionbut not the same sequence as the miR34a-antagomir; BPD:hyperoxia-exposed mouse model at PN14.

FIG. 6 is a blot showing protein expression of Ang1, Notch2, and Sirt1in the mice lungs administered miR34a-antagomir in RA, at PN14, alongwith appropriate control groups. RA: room air; Scrambled: (control) RNAwith the same nucleotide composition but not the same sequence as themiR34a-antagomir; BPD: hyperoxia-exposed mouse model at PN14.

FIG. 7 is a panel of images showing lung histology and morphometry innewborn WT mice administered miR34a inhibitor in the BPD mouse model atPN14. Scrambled (Scr) or miR34a inhibitor was delivered intranasally onPN1, with repeat doses on PN3 and PN4. Control mice were kept in RA frombirth until PN14. Representative photomicrographs of mouse lungs (H&Estain) at PN14 are shown at low-magnification (10×: top panels) andhigh-magnification (20×: bottom panels).

FIG. 8 is a graph showing chord length measurements in the mice lungsadministered miR34a-inhibitor in the hyperoxia-induced murine BPD model,at PN14, along with appropriate control groups.

FIG. 9 is a panel of images showing showing lung histology andmorphometry in newborn WT mice and IFN-γ transgenic mice (IFN-γ TG)treated with water or doxicycline (DOX) to induce expression of IFN-γwhich mimics the BPD mouse model at PN14 as well as human BPD.

FIG. 10A is a graph showing increased levels of IFN-γ in IFN-γ TG micetreated with DOX to induce expression of IFN-γ as compared to WT micetreated with water or DOX or IFN-γ TG mice treated with water.

FIG. 10B is a graph showing chord length in in IFN-γ TG mice treatedwith DOX to induce expression of IFN-γ as compared to WT mice treatedwith water or DOX or IFN-γ TG mice treated with water.

FIG. 11 is a panel of images showing lung histology and morphometry innewborn WT mice and TGFβ1 transgenic mice (TGFβ1 TG) treated with wateror DOX to induce expression of TGFβ1 which mimics the BPD mouse model atPN14 as well as human BPD.

FIG. 12A is a graph showing increased levels of miR34a expression in thelungs of TGFβ1 TG mice treated with DOX to induce expression of TGFβ1 ascompared to WT mice treated with water or DOX or TGFβ1 TG mice treatedwith water.

FIG. 12B is a Western Blot showing that the expression of Ang1 isdecreased in the TGFβ1 TG mice treated with DOX to induce expression ofTGFβ1

FIG. 13 is a panel of images showing the effect of surfactant on siRNAtransfection. Representative fluorescence microscopy images are shownfor (1) untreated, (2) scrambled siRNA, (3) siRNA and lipid transfectionreagent (Lipofectamine 2000®), (4) siRNA and 5 μL surfactant(Curosurf®), (5) siRNA and 10 μL surfactant (Curosurf®), and (6) siRNAand 20 μL surfactant (Curosurf®). Red areas indicate presence of siRNA.

FIG. 14 is a graph showing transfection ratios for each group. Each barrepresents the mean±the SEM for a minimum of 3 samples per group.*P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001, unpaired t-test.(Curo=Curosurf®, Lipo=Lipofectamine 2000®).

FIG. 15 is a Western Blot showing the in vitro effect of surfactant(Curosurf®) as a transfection reagent on C/EBP homologous protein (CHOP;also known as growth arrest and DNA damage-inducible gene 153/GADD153)expression in hyperoxic exposure. The protein expression of CHOP inMLE-12 cells was ascertained under the conditions described, usingβ-actin as a control.

FIG. 16 is a graph showing the corresponding relative CHOP proteinexpression in MLE-12 cells. Each bar represents the mean±the SEM for aminimum of 3 samples per group. *P≦0.05, **P≦0.01, unpaired t-test.(Curo=Curosurf®, Hyp=hyperoxia, Lipo=Lipofectamine 2000®, siCHOP=CHOPsiRNA).

FIG. 17 is a Western Blot showing CHOP expression after siRNA wasdelivered intranasally on PN1 and PN3 to newborn WT mice (C57Bl6/Jstrain) during room air (RA) or hyperoxia (HYP) exposure for 4 days. Inthe hyperoxia-exposed mice, an additional group was administered CHOPsiRNA with surfactant (Curosurf®) as the delivery vehicle. Thespecificity of the CHOP siRNA-induced decrease in CHOP proteinexpression, with surfactant (Curosurf®) as the delivery vehicle inhyperoxia, was confirmed by using scrambled siRNA controls

FIG. 18 is a Western Blot showing CHOP protein expression after CHOP orscrambled siRNA was delivered intranasally with surfactant (Curosurf®)as the delivery vehicleon PN1 and PN3 to newborn WT mice (C57B16/Jstrain) during room air (RA) or hyperoxia (HYP) exposure for 4 days.β-actin expression was used as a control.

FIG. 19A is a panel of images showing lung histology in newborn WT miceadministered CHOP siRNA alone or with surfactant (Curosurf®) as thedelivery vehicle in the BPD mouse model at PN14. CHOP siRNA with orwithout surfactant was delivered intranasally on PN1, with repeat doseson PN3 and PN4. Control mice were kept in RA from birth until PN14.Representative photomicrographs of mouse lungs (H&E stain) at PN14 areshown at low-magnification (10×).

FIG. 19B is a graph showing lung morphometry (chord length) in newbornWT mice administered CHOP or scrambled siRNA alone or with surfactant(Curosurf®) as the delivery vehicle in the BPD mouse model at PN14.Scrambled or CHOP siRNA was delivered intranasally on PN1, with repeatdoses on PN3 and PN4. Control mice were kept in RA from birth untilPN14. *P≦0.05, **P≦0.01. RA: room air; siCHOP: CHOP siRNA.

FIG. 20 is a Western Blot showing the in vitro effect of surfactant(Curosurf®) as a transfection reagent on Angiopoietin 2 (Ang2)expression in hyperoxic exposure. The protein expression of Ang2 inMLE-12 cells was ascertained under the conditions described, using βactin as a control. (Curo=Curosurf®, siAng2=Ang2 siRNA).

FIG. 21A is a panel of images showing lung histology in newborn WT miceadministered Ang2 siRNA alone or with surfactant (Curosurf®) as thedelivery vehicle in the BPD mouse model at PN14. Ang2 siRNA with orwithout surfactant was delivered intranasally on PN1, with repeat doseson PN3 and PN4. Control mice were kept in RA from birth until PN14.Representative photomicrographs of mouse lungs (H&E stain) at PN14 areshown at low-magnification (10×).

FIG. 21B is a graph showing lung morphometry (chord length) in newbornWT mice administered Ang2 siRNA alone or with surfactant (Curosurf®) asthe delivery vehicle in the BPD mouse model at PN14. Ang2 siRNA wasdelivered intranasally on PN1, with repeat doses on PN3 and PN4. Controlmice were kept in RA from birth until PN14. *P≦0.05, **P≦0.01. RA: roomair; Curo=Curosurf®; siAng2: Ang2 siRNA.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein may be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

As used herein, the articles “a” and “an” are used to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein when referring to a measurable value such as an amount, atemporal duration, and the like, the term “about” is meant to encompassvariations of ±20% or within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5%, 0.1%, 0.05%, or 0.01% of the specified value, as such variationsare appropriate to perform the disclosed methods. Unless otherwise clearfrom context, all numerical values provided herein may be modified bythe term about.

By “alteration” is meant a change (increase or decrease) in theexpression levels or activity of a gene or polypeptide as detected bystandard art known methods such as those described herein. As usedherein, an alteration includes a 10% change in expression levels,preferably a 25% change, more preferably a 40% change, and mostpreferably a 50% or greater change in expression levels.”

By “alveoli” is meant a cellular air sac at the terminal end of therespiratory tract. Epithelial cells, such as type I and type II alveolarcells, form the structure of the alveoli and are interspersed withcapillaries for gas exchange.

By “antagomir” is meant anti-miRs or blocking oligonucleotides thatprevent other molecules from binding a mRNA molecule. Antagomirs aresmall synthetic RNAs that are complementary to a miRNA target to silenceendogenous miRNA. The antagomir may include modifications to preventdegradation and/or mispairings to prevent enzymatic cleavage. In oneembodiment, the antagomir is a miR34a antagomir.

By “biomarker” or “marker” is meant any protein or polynucleotide havingan alteration in expression level or activity that is associated with adisease or disorder.

By “bronchopulmonary dysplasia” or “BPD” is meant a chronic lungcondition, disorder or disease that most commonly affects infants andchildren, characterized by abnormal alveolarization or injury tomicrovascularture that results in reduction of overall surface area forsufficient gas exchange.

By “complementary sequence” or “complement” is meant a nucleic acid basesequence that can form a double-stranded structure by matching basepairs to another polynucleotide sequence. Base pairing occurs throughthe formation of hydrogen bonds, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleobases.For example, adenine and thymine are complementary nucleobases that pairthrough the formation of hydrogen bonds.

By “disorder” is meant any condition or disease that damages orinterferes with the normal function of a cell in a lung, a lung tissue,or an organ of the respiratory system. Examples of disorders includerespiratory distress syndrome (RDS), tachypnea, tachycardia, frequentdesaturations, pulmonary hypoplasia, dysregulated vascularization, orthe like.

By “dysregulated vascularization” is meant dysfunctional, abnormal orinhibition of blood vessels or capillaries formation in the lung, suchas the alveoli.

By “effective amount” is meant the amount required to reduce or improveat least one symptom of a disease relative to an untreated patient. Theeffective amount of active compound(s) used to practice the presentinvention for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

By “fragment” is meant a portion of a polynucleotide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acids. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80,90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000 or2500 (and any integer value in between) nucleotides. The fragment, asapplied to a nucleic acid molecule, refers to a subsequence of a largernucleic acid. A “fragment” of a nucleic acid molecule may be at leastabout 15 nucleotides in length; for example, at least about 50nucleotides to about 100 nucleotides; at least about 100 to about 500nucleotides, at least about 500 to about 1000 nucleotides, at leastabout 1000 nucleotides to about 1500 nucleotides; or about 1500nucleotides to about 2500 nucleotides; or about 2500 nucleotides (andany integer value in between).

By “hyperoxic” or “hyperoxia” is meant an excess of oxygen or higherthan normal partial pressure (atmospheric) of oxygen. Hyperoxia in thelung can lead to oxygen toxicity, cellular damage, and lung injury.

By “hyperoxia-induced gene” is meant a gene whose expression is induced,either directly or indirectly, under hyperoxic conditions. Examples ofsuch hyperoxia-induced genes include, but are not limited to C/EBPhomologous protein (CHOP), interferon-gamma (IFN-γ), transforming growthfactor-beta 1 (TGF-β1), and angiopoietin 2 (Ang2), and anti-inflammatoryproteins such as Sirt1, Bcl2, Tie2, Akt, DLL1, Notch 1, Notch 2, CDK4,Cyclin D1, caspase 3, caspase 8, caspase 9, Fas, and Fas-L, or anycombination thereof.

As used herein, the term “inhibit” is meant to refer to a decrease inbiological state. For example, the term “inhibit” may be construed torefer to the ability to negatively regulate the expression, stability oractivity of a protein, including but not limited to transcription of aprotein mRNA, stability of a protein mRNA, translation of a proteinmRNA, stability of a protein polypeptide, a protein post-translationalmodifications, a protein activity, a protein signaling pathway or anycombination thereof.

Further, the term “inhibit” may be construed to refer to the ability tonegatively regulate the expression, stability or activity of a miRNA,wherein such inhibition of the miRNA may affect modulation of a gene,protein mRNA, stability of a protein mRNA, translation of a proteinmRNA, stability of a protein, a protein post-translationalmodifications, and/or a protein activity.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionthat may be used to communicate the usefulness of the compounds of theinvention. In some instances, the instructional material may be part ofa kit useful for effecting alleviating or treating the various diseasesor disorders recited herein. Optionally, or alternately, theinstructional material may describe one or more methods of alleviatingthe diseases or disorders in a cell or a tissue of a mammal. Theinstructional material of the kit may, for example, be affixed to acontainer that contains the compounds of the invention or be shippedtogether with a container that contains the compounds. Alternatively,the instructional material may be shipped separately from the containerwith the intention that the recipient uses the instructional materialand the compound cooperatively. For example, the instructional materialis for use of a kit; instructions for use of the compound; orinstructions for use of a formulation of the compound.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation. A “purified” or“biologically pure” protein is sufficiently free of other materials suchthat any impurities do not materially affect the biological propertiesof the protein or cause other adverse consequences. That is, a nucleicacid or peptide of this invention is purified if it is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Purity and homogeneity aretypically determined using analytical chemistry techniques, for example,polyacrylamide gel electrophoresis or high performance liquidchromatography. The term “purified” can denote that a nucleic acid orprotein gives rise to essentially one band in an electrophoretic gel.For a protein that can be subjected to modifications, for example,phosphorylation or glycosylation, different modifications may give riseto different isolated proteins, which can be separately purified.

By “microRNA” or “miRNA” or “miR” is meant a small non-coding RNA, whichfunctions in transcriptional and/or post-transcriptional regulation ofgene expression.

By “non-polymeric” is meant to lack a fixed or crosslinked polymernetwork made up of synthetic or biomolecular repeating units. In oneembodiment, a composition is non-polymeric when it lacks a fixed orcrosslinked polymer network, such as lacking a hydrogel, nano- ormicro-particle or gel, nano- or micro-molecule, or similar component.

“Pharmaceutically acceptable” refers to those properties and/orsubstances that are acceptable to the patient from apharmacological/toxicological point of view and to the manufacturingpharmaceutical chemist from a physical/chemical point of view regardingcomposition, formulation, stability, patient acceptance andbioavailability. “Pharmaceutically acceptable carrier” refers to amedium that does not interfere with the effectiveness of the biologicalactivity of the active ingredient(s) and is not toxic to the host towhich it is administered.

As used herein, the term “pharmaceutical composition” or“pharmaceutically acceptable composition” refers to a mixture of atleast one compound or molecule useful within the invention with apharmaceutically acceptable carrier. The pharmaceutical compositionfacilitates administration of the compound or molecule to a patient.Multiple techniques of administering a compound or molecule exist in theart including, but not limited to, intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound or molecule usefulwithin the invention within or to the patient such that it may performits intended function. Typically, such constructs are carried ortransported from one organ, or portion of the body, to another organ, orportion of the body. Each carrier must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation,including the compound useful within the invention, and not injurious tothe patient. Some examples of materials that may serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; surface activeagents; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations. As usedherein, “pharmaceutically acceptable carrier” also includes any and allcoatings, antibacterial and antifungal agents, and absorption delayingagents, and the like that are compatible with the activity of thecompound useful within the invention, and are physiologically acceptableto the patient. Supplementary active compounds may also be incorporatedinto the compositions. The “pharmaceutically acceptable carrier” mayfurther include a pharmaceutically acceptable salt of the compound ormolecule useful within the invention. Other additional ingredients thatmay be included in the pharmaceutical compositions used in the practiceof the invention are known in the art and described, for example inRemington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co.,1985, Easton, Pa.), which is incorporated herein by reference.

By “phospholipid” is meant a molecule with a hydrophilic phosphate groupconnected to a hydrophobic lipid. Examples of a phospholipid include,but are not limited to, phosphotidylcholine, phosphatidic acid,phosphatidylethanolamine, phosphatidylserine, a phosphoinositide such asphosphatidylinositol, phosphatidylinositol phosphate, aphosphosphingolipid such as ceramide phosphorylcholine, ceramidephosphorylethanolamine, and ceramide phosphoryllipid, or any derivativethereof.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which may be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides may be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences that are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means. The following abbreviations for thecommonly occurring nucleic acid bases are used. “A” refers to adenosine,“C” refers to cytosine, “G” refers to guanosine, “T” refers tothymidine, and “U” refers to uridine. The term “RNA” as used herein isdefined as ribonucleic acid. The term “recombinant DNA” as used hereinis defined as DNA produced by joining pieces of DNA from differentsources.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) thatis free of the genes which, in the naturally-occurring genome of theorganism from which the nucleic acid molecule of the invention isderived, flank the gene. The term therefore includes, for example, arecombinant DNA that is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic DNA of a prokaryote oreukaryote; or that exists as a separate molecule (for example, a cDNA ora genomic or cDNA fragment produced by PCR or restriction endonucleasedigestion) independent of other sequences. In addition, the termincludes an RNA molecule that is transcribed from a DNA molecule, aswell as a recombinant DNA that is part of a hybrid gene encodingadditional polypeptide sequence.

As used herein, the terms “prevent,” “preventing,” “prevention,” and thelike refer to reducing the probability of developing a disorder orcondition in a subject, who does not have, but is at risk of orsusceptible to developing a disorder or condition.

By “reduces” or “decreases” is meant a negative alteration of at least10%, 25%, 50%, 75%, or 100%.

By “reference” is meant a standard or control. A “reference” is also adefined standard or control used as a basis for comparison.

As used herein, “sample” or “biological sample” refers to anything,which may contain the biomarker (e.g., polypeptide, polynucleotide, orfragment thereof) for which a biomarker assay is desired. The sample maybe a biological sample, such as a biological fluid or a biologicaltissue. In one embodiment, a biological sample is a tissue sampleincluding pulmonary vascular cells. Such a sample may include diversecells, proteins, and genetic material. Examples of biological tissuesalso include organs, tumors, lymph nodes, arteries and individualcell(s). Examples of biological fluids include urine, blood, plasma,serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,mucus, amniotic fluid or the like.

By “small interfering RNA” or “siRNA” is meant a short RNA molecule thatmay be double stranded, which interferes with the expression of aspecific gene that includes a nucleotide sequence complementary to theRNA molecule.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

A “subject” or “patient,” as used therein, may be a human or non-humanmammal. Non-human mammals include, for example, livestock and pets, suchas ovine, bovine, porcine, canine, feline and murine mammals.Preferably, the subject is human.

By “surfactant” is meant a compound that lowers the surface tensionbetween two liquids or a liquid and a solid. The surfactant is acompound that is amphiphilic with a water soluble component and a waterinsoluble component.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or improving a disorder and/or symptom associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely ameliorated or eliminated.

A “vector” is a composition of matter that comprises an isolated nucleicacid and that may be used to deliver the isolated nucleic acid to theinterior of a cell. Numerous vectors are known in the art including, butnot limited to, linear polynucleotides, polynucleotides associated withionic or amphiphilic compounds, plasmids, and viruses. Thus, the term“vector” includes an autonomously replicating plasmid or a virus. Theterm should also be construed to include non-plasmid and non-viralcompounds which facilitate transfer of nucleic acid into cells, such as,for example, polylysine compounds, liposomes, and the like. Examples ofviral vectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, and the like.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression may be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, partial integers, orsub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, or 50.

The recitation of an embodiment for a variable or aspect herein includesthat embodiment as any single embodiment or in combination with anyother embodiments or portions thereof. Any compositions or methodsprovided herein can be combined with one or more of any of the othercompositions and methods provided herein.

Compositions

Bronchopulmonary dysplasia and related conditions and disease, such ashyperoxia, result in inflammation, dysregulated vascularization,impaired alveolarization, and injurious cell death. Blocking thedeleterious effects of genes involved in inflammation, cell death anddysregulated cell proliferation is one mechanism to treat or prevent oneor more symptoms of bronchopulmonary dysplasia. Compositions and methodsare included herein for effective and efficient gene inhibition in alung.

The invention includes, in one aspect, a non-polymeric compositioncomprising a small interfering RNA (siRNA) capable of inhibitingexpression of a gene, and a surfactant, wherein the composition isformulated for delivery to a lung tissue. A non-polymeric compositionlacks a fixed or crosslinked polymeric component. For example, thenon-polymeric composition lacks a hydrogel, nano- or micro-particle orgel, nano- or micro-molecule, or similar component.

In one embodiment, the siRNA comprises an RNA that inhibits expressionof at least one gene encoding a protein selected from the groupconsisting of C/EBP homologous protein (CHOP), interferon-gamma (IFN-γ),transforming growth factor-beta 1 (TGF-β1), and angiopoietin 2 (Ang2).For example, CHOP siRNA: UCAAGAGUAGUGAAGGUUUTT; Ang2 siRNA:UUGUCGUCUGGUUUAGUACTT; Anti-sense made against the mature miRNA34asequence 5′-UGGCAGUGUCUUAGCUGGUUGU-3′. In yet another embodiment, thesiRNA is an antagomir, such as a miR34a antagomir. In still yet anotherembodiment, siRNA comprises an RNA that inhibits expression of at leastone gene encoding an anti-inflammatory protein selected from the groupconsisting of Sirt1, Bcl2, Ang1, Tie2, Akt, DLL1, Notch 1, Notch 2,CDK4, Cyclin D1, caspase 3, caspase 8, caspase 9, Fas, and Fas-L.

To enhance delivery of the siRNA or increase transfection/uptakeefficiency of the siRNA into lung tissue to inhibit gene expression, thecomposition also includes a surfactant. The surfactant can serve as avehicle to deliver or administer the siRNA to the lung. The surfactantcan include a phospholipid, such as phosphotidylcholine and derivativesthereof.

As it is desirable to deliver the composition to the lung, in oneembodiment, the composition is formulated for intranasal delivery. Inanother embodiment, the composition is formulated for inhalationdelivery. The composition may be aerosolized for enhanced delivery andadministration.

The composition may further include additional agents and inhibitors ofgene expression. In one embodiment, the composition further comprises aninhibitor of cox-2.

Pharmaceutical Compositions

The invention also encompasses the use of a pharmaceutical compositionof the invention to practice the methods of the invention. Such apharmaceutical composition may be provided in a form suitable foradministration to a subject, and may be comprise one or morepharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The at least one compositionof the invention may comprise a physiologically acceptable salt, such asa compound contemplated within the invention in combination with aphysiologically acceptable cation or anion, as is well known in the art.

Pharmaceutical compositions that are useful in the methods of theinvention may be suitably developed for inhalational, oral, rectal,vaginal, parenteral, topical, transdermal, pulmonary, intranasal,buccal, ophthalmic, intrathecal, intravenous or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations. Theroute(s) of administration will be readily apparent to the skilledartisan and will depend upon any number of factors including the typeand severity of the disease being treated, the type and age of theveterinary or human patient being treated, and the like.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

In one embodiment, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inone embodiment, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of at least one compound ofthe invention and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers, which are useful, include, but arenot limited to, glycerol, water, saline, ethanol and otherpharmaceutically acceptable salt solutions such as phosphates and saltsof organic acids. Examples of these and other pharmaceuticallyacceptable carriers are described in Remington's Pharmaceutical Sciences(1991, Mack Publication Co., New Jersey).

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook,2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of AnimalCells” (Freshney, 2010); “Methods in Enzymology” “Handbook ofExperimental Immunology” (Weir, 1997); “Gene Transfer Vectors forMammalian Cells” (Miller and Calos, 1987); “Short Protocols in MolecularBiology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles,Applications and Troubleshooting”, (Babar, 2011); “Current Protocols inImmunology” (Coligan, 2002). These techniques are applicable to theproduction of the polynucleotides and polypeptides of the invention,and, as such, may be considered in making and practicing the invention.Particularly useful techniques for particular embodiments will bediscussed in the sections that follow.

Method of Treatment

The present invention also includes methods of treating a lung in asubject in need thereof, such as a subject with bronchopulmonarydysplasia. In one aspect, a method includes inhibiting gene expressionin a lung of a subject in need thereof comprising administering atherapeutically effective amount of a non-polymeric composition to thelung of the subject, wherein the non-polymeric composition comprises asmall interfering RNA (siRNA) capable of inhibiting expression of agene, and a surfactant.

In another aspect, a method includes treating bronchopulmonary dysplasiain a lung of a subject comprising administering a therapeuticallyeffective amount of a non-polymeric composition to the lung of thesubject, wherein the non-polymeric composition comprises a smallinterfering RNA (siRNA) capable of inhibiting expression of ahyperoxia-induced gene, and a surfactant.

In one embodiment, the siRNA inhibits gene expression of at least oneanti-inflammatory molecule. The anti-inflammatory molecule can beselected from the group consisting of Sirt1, Bcl2, Ang1, Tie2, Akt,DLL1, Notch 1, Notch 2, CDK4, Cyclin D1, caspase 3, caspase 8, caspase9, Fas, and Fas-L. In another embodiment, the siRNA inhibits geneexpression of hyperoxia-induced gene, an anti-inflammatory molecule, orother molecule such as C/EBP homologous protein (CHOP), interferon-gamma(IFN-γ), transforming growth factor-beta 1 (TGF-β1), angiopoietin 2(Ang2), or any combination thereof. In another embodiment, the siRNAinhibits gene expression of at least one molecule selected from thegroup consisting of C/EBP homologous protein (CHOP), interferon-gamma(IFN-γ), transforming growth factor-beta 1 (TGF-β1), and angiopoietin 2(Ang2).

When the composition is administered to the lung, the composition isformulated for such administration. In one embodiment, theadministration delivers the composition to alveoli in the lung. Inanother embodiment, the composition is formulated for inhalationadministration or intranasal administration. In some embodiment, thelung is hyperoxic, such as in a subject with bronchopulmonary dysplasiaor hyperoxia-induced cell death in the lung.

In another embodiment, the method further comprises administering aninhibitor of cox-2. In yet another embodiment, the method furthercomprises assessing dysregulated vascularization in the lung.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

Examples

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out embodiments of the presentinvention, and are not to be construed as limiting in any way.

The Materials and Methods used in the performance of the experimentsdisclosed herein are now described.

Transgenic Mice

C57BL6/J mice were uses in the experimental studies. IFNγ overexpressingtriple transgenic (TTG) CC10-rtTA/tTs-IFNγ mice were generated in thelaboratory (Am J Respir Cell Mol Biol. 2011 May; 44(5):621-30. Epub 2011Jan. 7). TGF-β1 transgenic (TG) mice had “human” TGF-β1 targeted to thelung using the CC10 promoter and was “turned on” with maternal exposureto doxycycline (dox) in the drinking water, leading to transmammaryactivation in the TG (+) pups, as described previously (BMC Cell Biol.2011 Dec. 15; 12:54).

Dox Water Administration

Lactating dams of newborn mice were given either regular water or doxwater (0.5 g/L) from PN1 through PN7, which allowed transmammaryactivation of IFNγ in the newborn lung, until sacrificed. Both groupsi.e. WT and IFNγ TTG had dox water, and regular water controls.

Western Blot Analysis

CHOP and Ang2 protein was detected with β-actin as control, from lunglysates using Western analysis.

Histologic Analysis

Animals were euthanized, a median sternotomy was performed, and rightheart perfusion was accomplished with calcium and magnesium-free PBS toclear the pulmonary intravascular space. The heart and lungs were thenremoved en bloc, then fixed to pressure (25 cm) with neutral-buffered10% Formalin, fixed overnight in 10% Formalin, embedded in paraffin,sectioned at 5 μm, and stained. Hematoxylin and eosin stains wereperformed.

Morphometric Analysis

Alveolar size was estimated from the mean cord length of the airspace asdescribed previously (Am J Respir Cell Mol Biol. 2011 May; 44(5):621-30.Epub 2011 Jan. 7). At least four animals were studied at each time pointin the presence and absence of dox water. Chord length increases withalveolar enlargement.

Oxygen Exposure

For the exposure to hyperoxia (100% O₂), newborn mice (along with theirmothers) were placed in cages in an airtight Plexiglas chamber (55×40×50cm) as described previously (Am J Respir Cell Mol Biol. 2011 May;44(5):621-30. Epub 2011 Jan. 7). Exposure to oxygen was initiated on PN1of life. Two lactating dams were used. Mothers were alternated inhyperoxia and room air every 24 h. The litter size was kept limited to12 pups to control for the effects of litter size on nutrition andgrowth. Throughout the experiment, they were given free access to foodand water. Oxygen levels were constantly monitored by an oxygen sensorthat was connected to a relay switch incorporated into the oxygen supplycircuit. The inside of the chamber was kept at atmospheric pressure, andmice were exposed to a 12 h light-dark cycle.

Mouse Model of BPD

Newborn WT mice were exposed to hyperoxia, as described herein, fromPN1-4 (saccular stage of murine lung development) and allowed to recoverin room air for the next 10 days. Mice were sacrificed on PN14. NB WTmouse lungs at PN14 have the phenotype mimicking human BPD. For the Cox2inhibition experiments, NB WT mice were injected daily with thecelecoxib 20 mg/k/d for 14 days.

Preparation and Administration of CHOP siRNA, Ang2 siRNA and miR34aAntagomir with Surfactant (Curosurf®). as the Delivery Vehicle

For In Vitro Experiments:

For CHOP and Ang2 silencing in MLE12 cells, 2×10⁵ cells per well wereplated into six-well plates and grown overnight until they were 50-80%confluent. CHOP, Ang2 and scrambled siRNA were transfected bylipofectamine 2000 or given wth or without surfactant (Curosurf®). Cellswere used after 24 h for the hyperoxia experiments.

For In Vivo Experiments:

CHOP siRNA (SEQ ID NO:1, UCAAGAGUAGUGAAGGUUUTT), Ang2 siRNA (SEQ IDNO:2, UUGUCGUCUGGUUUAGUACTT) or miR34a-antagomir (SEQ ID NO:3,UGGCAGUGUCUUAGCUGGUUGU) was suspended in nuclease free water to make a20 μM final concentration. Surfactant (Curosurf®) was used as thedelivery vehicle in a volume of 5 μl. The newborn mouse was held by itsears and 1-2 μl per nostril of the mix was gradually released into thenostrils with the help of a long tipped micropipette. The rate ofrelease was adjusted so as to allow the mouse to inhale the solutionwithout trying to form bubbles. The mouse was held in the hangingposition for another couple of minutes till its breathing graduallyreturned to normal. The control mice were administered Scrambled siRNAof the same concentration intranasally at the same volume. The doses ofCHOP siRNA, Ang2siRNA or miR34a-antagomir and scrambled siRNA wereadministered on PN1 and PN3 in the mouse model of BPD.

Human Lung Samples of BPD

Human lung tissue samples were obtained postmortem from fetuses andpremature infants having the diagnoses of respiratory distress syndrome1-2 days (RDS 1-2), RDS 3-7 days (RDS 3-7), RDS>7 days (RDS>7), BPD andterm infants as controls.

The Results of the experiments disclosed herein are now described.

The miR profile of BPD remains largely unexamined. Because miRs regulatemultiple cellular functions critical for organ development and diseaseconditions, it is possible that miR could be a “master” regulator for acomplex disorder such as BPD. Recently, miR34a expression has been notedin the hyperoxia-exposed lungs of rat and mouse and in human asthma, acondition to which BPD patients appear to be predisposed long-term. Inaddition, miR34a has been detected in serum and other body fluids ofhumans, underscoring its potential as a diagnostic and prognosticbiomarker. miR34a has not been implicated in human BPD.

In the experiment illustrated in FIGS. 2A and 2B, a hyperoxia-inducedmouse model of BPD was used to carry out initial in vivo studies ofmiR34a. In this experiment, newborn mice pups were exposed to hyperoxia(100% oxygen) from postnatal stage PN1 to PN4, a timeframe thatcorresponds to the saccular stage of lung development and is equivalentto human premature infants at 23-28 weeks of gestation, when BNP beginsto develop. The mice were then allowed to recover in room air from PN5to PN14, which corresponds to the alveolar stage of development and isequivalent to human premature infants after 32-36 weeks of gestation, atwhich point the development of BPD was well underway. When mice weresacrificed at PN14, the lungs had a clearcut BPD phenotype. If the micewere allowed to live to adulthood, the pulmonary phenotype of BPDpersisted, mimicking the course of the disease in humans. In theseexperiments, a rapid induction in expression of the mature form ofmiR34a at PN4 was found, which increased 5-fold by PN7 (FIG. 1). FIG. 2Ais a panel of images showing the absence (left image) or presence (rightimage) of hyperoxia in a hyperoxia-induced BPD mouse model. FIG. 2B is agraph showing the cord length (an indicator of mean alveolar size) innewborn wild type (WT) mice or hyperoxia-induced BPD mice at post natalday 14 (PN14).

Preliminary studies characterizing the expression of miR34a in humanbabies suffering from BPD were also analyzed. Because many infantsurvivors of respiratory distress syndrome (RDS) later develop BPD,neonates with RDS were analyzed for elevated levels of miR34a. Toaccomplish this goal, quantitative real-time PCR was used to measuremiR34a expression in tracheal aspirate cell pellets from infants who hadRDS severe enough to require intubation and surfactant treatment duringthe first week of life (n=35; 550-1250 g birth weight). The resultsshowed a significant (p<0.001) increase in miR34a gene expression inthese samples (FIG. 3).

To determine localization of the increased miR34a expression in thelung, in situ hybridization was used to assess the expression of miR34ain lungs of human neonates at various stages of development with/withoutRDS and BPD. There was a substantial increase in miR34a staining in thelungs of premature infants with RDS and BPD, and it was localized toalveolar epithelial and inflammatory cells (FIG. 4). These data areconsistent with mouse models and support the idea that miR34a expressionin the lung during the neonatal period may have a role in BPDpathogenesis. Thus, miR34a could serve as a diagnostic/prognostic markerfor BPD and could also provide a therapeutic target for this disease.

To determine the role of miR34a in the development of BPD, wild-type(WT) mice were treated with miR34a-antagomir. Following treatment ofnormal mice in room air, chord length was measured. Longer chord lengthwas consistent with the disrupted alveolarization that is characteristicof BPD (FIG. 5). Decreased expression levels of known down-streamtargets of miR34a in the lungs of these mice were also found, includingthe anti-inflammatory molecule Sirtuin 1 (Sirt1), the vascular mediationAngiopoietin 1 (Ang1), and Notch2 (FIG. 6), providing further evidenceof BPD pathology.

In subsequent proof-of-principle studies, the sequence of miR34a wasused to generate an antisense oligonucleotide molecule, or antagomir,that inhibits miR34a. The dose was optimized to 100 nM based on in vitrotesting in MLE-cells for maximal down-regulation of miR34a and used thehyperoxia-induced mouse model of BPD to test the effect of themiR34a-inhibitor in vivo. The miR34a-inhibitor was given at post-natalstage PN1 and repeated at PN3 and PN4, and histological staining (FIG.7) and chord length measurements (FIG. 8) both showed that themiR34a-inhibitor restored the normal alveolarization in the BPD lungs.

The research has indicated that hyperoxia leads to the production andrelease of the mature form of miR34a. This release, in turn, increasesalveolarization and leads to decreased expression of downstream miR34atargets including Ang1 (decreased expression of which is known toincrease cell death in hyperoxia-induced lung injury and BPD models) andSirt1 (which has been associated with enhanced transcription ofproinflammatory mediators and BPD). The combined effect of enhanced celldeath and decreased cell proliferation would be impaired alveolarizationin the lung. Furthermore, it is hypothesized that miR34a enhances celldeath by suppressing the Notch signaling pathway, resulting indysregulated vascularization in the lung, one of the hallmarks of BPD.

Importantly, miRs are post-transcriptional regulators critical fororganogenesis and pathogenesis, and because a single miR can targetmultiple functionally-related genes, miR-based therapies have thepotential to be more effective than single gene approaches. For example,miR34a down-regulation (inhibition) decreases epithelial cell death (viaAng1) and inflammation (via Sirt1), while improving vascularization (viaNotch2). It seems reasonable to think that these effects, takentogether, will contribute to the recovery of epithelial cells andimproved alveolarization.

To determine what effects miR34a has on cell death, surfactant-enhanceddelivery of siRNA directed against miR34a is being tested in 2additional (to the hyperoxia-induced BPD model described above) modelsof BPD. The first is the interferon-gamma (IFN-γ) inducible transgenicmice which were treated with doxcycline (DOX) to induce IFN-γ. IFN-γinduction in newborn mice pups was from postnatal stage PN1 to PN7, atimeframe that corresponds to the saccular/early alveolar stage of lungdevelopment and is equivalent to human premature infants at 23-30 weeksof gestation, when BPD is most likely to develop. When mice weresacrificed at PN7, the lungs had a clearcut BPD phenotype, FIG. 9. Themice had increased levels of IFN-γ (FIG. 10A) and longer chord lengths(FIG. 10B), indicative of BPD.

The second is transforming growth factor beta 1 (TGFβ1) inducibletransgenic mice which were treated with DOX to induce TGFβ1. TGFβ1induction in newborn mice pups from postnatal stage PN7 to PN10, atimeframe that corresponds to the early alveolar stage of lungdevelopment and is equivalent to human premature infants at 28-32 weeksof gestation, when the process of developing BPD is well under way. Whenmice were sacrificed at PN10, the lungs had a clearcut BPD phenotype,FIG. 11. The lungs of these mice had increased levels of miR34aexpression (FIG. 12A) and decreased Ang1 expression (FIG. 12B),indicative of the potential role of miR34a in an additional mouse modelof BPD.

The biological activity and efficacy of siRNA targeted againstinterferon-gamma (IFN-γ), transforming growth factor-beta 1(TGF-β1) andAngiopoietin 2 (Ang2), using RNAse-free water or surfactant (Curosurf®)as the vehicle, in an in vitro system was also of interest. Anintranasal delivery of siRNA targeting IFN-γ, TGF-β1 and Ang2 to newborn(NB) mice lungs in the hyperoxia-induced mouse model of BPD and inlung-specific doxycycline-inducible IFN-γ, TGF-β1 and Ang2overexpressing transgenic (TG) mice was tested.

Utilizing developmentally-appropriate mouse models, a hyperoxia-inducedand lung-specific doxycycline-inducible IFN-γ and TGFβ1 TG BPD modelsdescribed above were used. Ang2 shows significant association with BPDin multiple independent cohorts of human BPD. Also a lung-specificdoxycycline-inducible model of Ang2 expression has a pulmonary phenotypecharacteristic of BPD.

Targeting these cytokines early in the course of the disease mayameliorate the lung phenotype of BPD. Efficacy of utilizing siRNAapproaches has been demonstrated in adult mice models. Experimentsdescribed herein demonstrate the feasibility of such an approach in thenewborn (NB) mouse lung.

For in vivo testing, a hyperoxia induced mouse model of BPD was used. Anoptimized dose of siRNA singly or in combination intranasally withsurfactant (such as Curosurf®) as the vehicle (or RNAse-free water ascontrol) was delivered to the NB mouse on postnatal (PN) days 1 and 3.Since the half-life of siRNA is ˜36 hours and in the model, thehyperoxia exposure is from PN1-4, this dosing protocol was sufficient.For the lung specific doxycycline-inducible models of IFN-γ, TGF-β1 andAng2, the dosing protocol was adjusted for administration on PN days 1,3, and 5. These NB mice specific induce gene expression with exposure todoxycycline on PN1, and by PN7 (at sacrifice), their lungs have the BPDphenotype. The efficacy of delivery and biological activity of theIFN-γ, TGF-β1 and Ang2 were evaluated by measuring mRNA and proteinexpression in the lung by real-time RT-PCR, western blot and doubleimmunohistochemical staining (using SP-B/C as the marker for Type IIpneumocytes) in the three models. The pulmonary phenotype was assessedby lung morphometry, bronchoalveolar lavage (BAL) cell counts, cytokineanalysis, histology, TUNEL assay (for cell death), and mRNA and proteinexpression of vascular and cell death mediators.

The phenomenon of gene silencing expression via RNA interference isdependent on effective transfection of siRNA. Hence, the efficacy ofsurfactant (such as Curosurf®) was assessed as a transfection reagentwith fluorescent-labeled scrambled siRNA. Other transfection reagents,such as Lipofectamine 2000®, was used as a positive control as it isknown to have a high transfection efficiency. MLE-12 cells, a pulmonaryadenocarcinoma cell line with alveolar ell characteristics, wereselected as a surrogate for alveolar epithelial cells.

As expected, immunofluorescent analysis revealed that detection ofscrambled siRNA within cells was dependent on the use of a transfectionreagent (FIG. 13). Lipofectamine 2000® had a transfection rate of 53%,over a 30-fold increase in efficiency when compared to siRNA alone(P≦0.0001; FIG. 14). Among the surfactant groups, cells treated with 10μL had the highest transfection rate at 4.5% (P≦0.0001; FIG. 14). Cellstreated with 20 μL of surfactant (Curosurf®) actually had a slightdecrease in transfection efficiency when compared to siRNA alone(P<0.05; FIG. 14). Taken together, these studies show that at properconcentrations Curosurf® is an enhancer of siRNA transfection inalveolar epithelial cells.

Hyperoxia exposure to the developing lung (in mouse and humans) leads toincreased IFN-γ levels and activation of the endoplasmic reticulum (ER)stress pathway; a key component of the ER stress pathway is the C/EBPhomologous protein (CHOP; also known as growth arrest and DNAdamage-inducible gene 153/GADD153). Elevated levels of CHOP areassociated with cell death via the ER stress-dependent pathway in thehyperoxia-induced model of BPD. To evaluate the use of surfactant(Curosurf®) to augment gene silencing using CHOP siRNA, CHOP proteinexpression was assessed by western blot analysis (FIG. 15). Exposure tohyperoxia resulted in an increase in CHOP when compared to room air.Both Lipofectamine 2000® and Curosurf® treated groups benefited from adecrease in CHOP protein expression when compared to untreated hyperoxiaand naked siRNA (P≦0.05; FIG. 16). There was no difference between theCurosurf® and Lipofectamine 2000® treated groups, suggesting comparableefficiency in gene silencing. Interestingly, there was no appreciabledifference between hyperoxia and naked siRNA groups. Thus, when used asa transfection reagent, Curosurf® serves as a potent enhancer of CHOPgene silencing via RNA interference.

The use of siRNA against the ER stress pathway mediator, CHOP, has beenshown to alleviate cell death in alveolar epithelial cells as well as inhyperoxia-induced and IFN-γ-mediated murine models of BPD. In addition,CHOP siRNA also restored alveolarization in the in vivo models. Hence,it was determined if surfactant-enhanced (Curosurf®) CHOP siRNA deliverywould further suppress CHOP protein expression in the hyperoxia-inducedmouse model of BPD. To test this, CHOP siRNA was delivered with orwithout surfactant, as the delivery vehicle, at postnatal (PN) day 1 and3, and the expression of CHOP protein was evaluated at PN4. The resultsare shown in FIGS. 17 and 18. The beneficial effects ofsurfactant-enhanced (Curosurf®) CHOP siRNA were confirmed with lunghistology and mophometry in the hyperoxia-exposed BPD model as shown inFIGS. 19A-19B.

Curosurf® also served as a potent enhancer of Ang2 gene silencing viaRNA interference in vitro and in vivo as noted in FIGS. 20 and 21A-21B,respectively. FIG. 20 shows similar efficacywith gene silencing withAng2 siRNA with or without surfactant. Surfactant-enhanced (Curosurf®)Ang2 siRNA delivery was significantly more effective than Ang2 siRNAalone in improving lung histology (FIG. 21A) and morphometry (chordlength; FIG. 21B). hyperoxia-induced mouse model of BPD.

In summary, FIGS. 13 and 14 established the optimal dosing regimen to beused when combining surfactant, such as Curosurf®, with siRNA. It isimportant to keep in mind that the transfection efficiency with lipidreagents, such as Lipofectamine, is exceedingly high, toxicity of suchagents have precluded them from being used in in vivo studies. FIGS. 15and 16 show that combining CHOP siRNA with surfactant has equivalentefficiency in suppressing CHOP expression as combining CHOP siRNA withLipofecatmine; both approaches being more efficient than naked CHOPsiRNA. Finally, as noted in FIG. 17, CHOP expression was markedlyincreased with hyperoxia exposure, and decreased with CHOP siRNAdelivery. When using surfactant as the delivery vehicle, there was afurther decrease in CHOP protein expression. Using surfactant as thedelivery vehicle did not negatively impact on the specificity of theresponse, as shown in FIG. 18. Surfactant-enhanced delivery of CHOP andAng2 improved the BPD lung architecture, with a more significant effectin the latter case, compared to the respective siRNAs alone, as shown inFIGS. 19 and 21A-21B.

Thus, it appears that surfactant, as the delivery vehicle, appears toenhance the effective delivery and distribution of specific siRNA invitro as well as in vivo in the lung, without impacting on thespecificity of the response.

Other Embodiments

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A non-polymeric composition comprising a smallinterfering RNA (siRNA) capable of inhibiting expression of a gene, anda surfactant, wherein the composition is formulated for delivery to alung tissue.
 2. The composition of claim 1, wherein the siRNA comprisesan RNA that inhibits expression of at least one gene encoding a proteinselected from the group consisting of C/EBP homologous protein (CHOP),interferon-gamma (IFN-γ), transforming growth factor-beta 1 (TGF-β1),and angiopoietin 2 (Ang2).
 3. The composition of claim 2, wherein thesiRNA is selected from the group consisting of a CHOP siRNA, an Ang2siRNA, and an anti-sense made against the mature miRNA34a sequence. 4.The composition of claim 2, wherein the siRNA is selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
 5. Thecomposition of claim 1, wherein the siRNA is an antagomir.
 6. Thecomposition of claim 5, wherein the siRNA is a miR34a antagomir.
 7. Thecomposition of claim 1, wherein the siRNA comprises an RNA that inhibitsexpression of at least one gene encoding an anti-inflammatory proteinselected from the group consisting of Sirt1, Bcl2, Ang1, Tie2, Akt,DLL1, Notch 1, Notch 2, CDK4, Cyclin D1, caspase 3, caspase 8, caspase9, Fas, and Fas-L.
 8. The composition of claim 1, wherein the surfactantcomprises a phospholipid.
 9. The composition of claim 8, wherein thephospholipid comprises phosphotidylcholine or derivatives thereof. 10.The composition of claim 1, wherein the composition is formulated forintranasal administration.
 11. The composition of claim 1, wherein thecomposition is formulated for administration by inhalation.
 12. Thecomposition of claim 1 further comprising an inhibitor of cox-2.
 13. Amethod of inhibiting gene expression in a lung of a subject in needthereof comprising: administering a therapeutically effective amount ofa non-polymeric composition to the lung of the subject, wherein thenon-polymeric composition comprises a small interfering RNA (siRNA)capable of inhibiting expression of a gene, and a surfactant.
 14. Themethod of claim 13, wherein the siRNA inhibits gene expression of atleast one anti-inflammatory molecule selected from the group consistingof Sirt1, Bcl2, Ang1, Tie2, Akt, DLL1, Notch 1, Notch 2, CDK4, CyclinD1, caspase 3, caspase 8, caspase 9, Fas, and Fas-L.
 15. The method ofclaim 13, wherein the siRNA inhibits gene expression of at least onemolecule selected from the group consisting of C/EBP homologous protein(CHOP), interferon-gamma (IFN-γ), transforming growth factor-beta 1(TGF-β1), and angiopoietin 2 (Ang2).
 16. The method of claim 13, whereinthe siRNA is selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, and SEQ ID NO:3.
 17. The method of claim 13, wherein theadministration delivers the composition to alveoli in the lung.
 18. Themethod of claim 13, wherein the composition is formulated for inhalationadministration or intranasal administration.
 19. The method of claim 13further comprising administering an inhibitor of cox-2.
 20. The methodof claim 13 further comprising assessing dysregulated vascularization inthe lung.
 21. The method of claim 13, wherein the lung is hyperoxic. 22.The method of claim 13, wherein the subject has bronchopulmonarydysplasia.
 23. The method of claim 13, wherein the subject hashyperoxia-induced cell death in the lung.
 24. A method of treatingbronchopulmonary dysplasia in a lung of a subject comprising:administering a therapeutically effective amount of a non-polymericcomposition to the lung of the subject, wherein the non-polymericcomposition comprises a small interfering RNA (siRNA) capable ofinhibiting expression of a hyperoxia-induced gene, and a surfactant.